This invention describes improved methods for derivatization and solubilization of fullerenes.
Since fullerenes were first macroscopically produced by the arc method in 1990 (Krätschmer, 1990), their exohedral derivatization chemistry has proceeded. The organic functionalization of fullerenes has been extensively reviewed (Hirsch, 1994(a); Wilson, 2000(a, b)) as well as their inorganic/organometallic functionalization chemistry (Balch, 1998). Because fullerenes possess no substituents, their exohedral derivatization chemistry begins exclusively with addition reactions to their carbon surface, most commonly by 1,2 bis-addition across the reactive carbon-carbon double bonds of the fullerene.
A fundamental characteristic of fullerenes is their electrophilic nature (Reed, 2000) and as a consequence most reported derivatization involves the addition of nucleophilic reagents. A particular kind of nucleophilic addition of widely recognized utility is cyclopropanation which was first reported by Bingel (Bingel, 1993). A general example of this so-called “Bingel” reaction is illustrated by the series of steps shown below in FIG. 1.
Many scientific papers and reports concern the Bingel-type cyclopropanation of the fullerene surface (Bingel, 1993; Bingel, 1995; Bingel, 1998 as well as PCT-WO96/09275A1, EP00695287B1, and EP00782560A1; Brettreich, 1998; Camps; 1997; Hirsch, 1994(a); Hirsch, 1994(b); Lamparth, 1994; Lamparth, 1997; Nierengarten, 1997; Richardson, 2000; Wei, 2001; Wharton, 2001; Wilson 2000(b)). A primary reference for general organic reactions between stabilized nucleophiles and electron-deficient alkenes (non-fullerenes) is that of Jung (1991).
The earliest examples of Bingel-type cyclopropanation of the fullerene surface involved base-induced deprotonation of an α-halo ketone, forming a relatively stabilized nucleophilic carbanion that attacks the electron deficient fullerene. Bingel's initial study employed NaH as the base, which forms gaseous hydrogen by combination of the metal hydride and the removed proton (Step 1). The incipient Na+ salt of the carbanion may be very strongly ion paired in the nonpolar solvents typically used (toluene, etc.). The carbanion may be partially stabilized by adjacent electron withdrawing substituents (the α-halogen, carbonyls, phenyls, etc. that are electronegative and/or inductively withdraw electron density) that simultaneously may or may not additionally enhance stability via electron delocalization (resonance).
The nucleophilic carbanion immediately attacks the fullerene, forming a new bond (Step 2). The cyclopropanation is complete following the spontaneous elimination of the halide anion (or other current leaving group) (Step 3). The net reaction thus occurs via an addition/elimination mechanism. While the mechanism illustrated in FIG. 1 is believed to be the most likely mechanism for the reaction, less-likely alternatives exist including a concerted reaction and an electron transfer route. By using excess quantities of reagents, multiple groups can be added to the fullerene surface. The different products with different numbers of groups added and with different isomeric arrangements on the fullerene surface can then be purified and separated from one another by chromatography. The fullerenes derivatized by cyclopropanation can also be referred to as methanofullerenes. When the Bingel reaction is performed on fullerenes that are already derivatized, for example C60F18, the α-halo substituent of the nucleophile may not function as the leaving group. Instead, a group previously on the fullerene departs leaving the nucleophile group attached to the fullerene via the α-sp3 carbon without cyclopropanation (Wei, 2001).
Conditions alternative to those used by Bingel in his first report have been developed. One of the most popular changes has been the replacement of the insoluble base NaH with soluble amines, most notably DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) (Camps, 1997). While this base is strongly hindered, it has been reported to readily form covalent bonds with the surface of C60 (Skiebe, 1994; Klos, 1994).
Another modification to the standard Bingel conditions reported is the incipient production of the reactive α-halo compounds by in situ treatment of mono- and bis-malonates with halogen-releasing agents such as CBr4, I2, etc. (Camps, 1997; Nierengarten, 1997). This allows for the use of more elaborately substituted malonates for which the α-halo precursor is difficult to individually prepare and/or isolate as a reagent. Bingel-style addition of malonates has been used to link fullerenes to a variety of substituents of interest, for example, porphyrins.
Addition of multiple groups to fullerene surfaces is usually performed stepwise using excess reagents. Derivatives with specific numbers of addends can be separated from derivatives with lesser or greater numbers of addends by chromatographic or other standard techniques. Separation of the different regioisomers formed can also be performed chromatograpically, but such separations can be expensive and laborious.
For a Bingel-style multi-derivatization, a tether-directed addition strategy offers control over regiospecificity of addition (location pattern of the cyclopropanations) and the number of addend groups in a pre-determined way while increasing yields over traditional derivatization strategies, as reviewed by Diederich et. al. (Diederich, F.; Kessinger, R. (1999). “Templated Regioselective and Stereoselective Synthesis in Fullerene Chemistry,” Acc. Chem. Res., 32, 537-545). The tether strategy uses multi-functional reaction substrates cojoined in one molecule by covalent moieties of variable length separating these functionalities, which are themselves variable in number.
Tether-directed formation of C60 trisadducts has been reported previously by Rapenne et. al. (Rapenne, G.; Crassous, J.; Collet, A.; Echegoyen, L.; Diederich, F. (1999). “Regioselective one-step synthesis of trans-3, trans-3, trans-3 and e, e, e [60]fullerene tris-adducts by a C3-symmetrical cyclotriveratrylene tether,” J. Chem. Soc. Chem. Commun. 1121-1122). Reuther et al. describe the use of cyclo-[n]-octylmalonates as an improved tether strategy (Reuther, U.; Brandmüller, T.; Donaubauer, W.; Hampel, F.; Hirsch, A. (2002). “A Highly Regioselective Approach to Multiple Adducts of C60 Governed by Strain Minimization of Macrocyclic Malonate Addends,” Chem. Eur. J., 8, 2261-2273). Reuther et. al. used this Bingel-reaction based tether addition process to form the C3 isomer (or e, e, e isomer) of the trisadduct C60[C(COOH)2]3 in high yield with high specificity. Bingel-style addition of malonates has also been used to control the regiochemistry of addition by Wilson (Wilson, 2000(b)).
Following the Bingel cyclopropanation, the ester groups of malonate addition products can be cleaved and converted to other functionalities, i.e. effecting side-chain modification (Lamparth, 1997). For example, the ester groups of the Bingel addition product of diethylbromomalonate (FIG. 1) can be converted to their respective carboxylic acids (or carboxylate salts of alkali metals, etc.) using the method of Hirsch (Lamparth, 1994). The carboxylic acid and carboxylate salts (of multiple adducts) of the fullerenes are water-soluble, a property critical to the development of the fullerenes' emerging medical applications. For example, multiply carboxylated C60 has shown high potential as an antioxidant for treating neurodegenerative disorders in vivo (Dugan, 2000). Other potential pharmaceutical applications for water-solubilized fullerenes include HIV-protease inhibitors (Wilson, 2000(a)), nuclear medicine agents (Cagle, 1999), and in vivo enhancers for medical imaging techniques including, e.g., MRI, X-ray, and nuclear imaging (Zhang, 1997; Wilson, 1999; Mikawa, 2001).
Typically, the Bingel-style cyclopropanation reactions of non-derivatized fullerenes have been conducted in non-polar solvents such as aromatic hydrocarbons (benzene, toluene, etc.) that are also good solvents for common fullerenes (Ruoff, 1993). The fullerenes derivatized in this manner have invariably belonged to the class of fullerenes possessing larger HOMO-LUMO gaps such as the most abundant fullerene, C60, and the larger “higher” C2n fullerenes, e.g. C70, C76, C78, C82, C84, etc. A notable property of these large HOMO-LUMO gap-possessing fullerenes is solubility in common non-polar organic solvents. Consequently, addition reactions like the Bingel method outlined above are performed on solutions of these fullerenes. This excludes important classes of fullerene materials that are not soluble in these common organic solvents. U.S. Pat. No. 5,739,376, issued to Bingel, reports formation of cyclopropanated derivatives of fullerenes in polar solvents including methylene chloride and chlorobenzene. The fullerenes discussed in Bingel's patent (C60, C70, C76, C78) are expected to be soluble in these polar aprotic solvents prior to derivatization. PCT published application WO 96/09275 of Bingel also reports the generation of fullerene derivatives by certain cyclopropanation reactions.
Chemical and electrochemical retro-Bingel reactions have been demonstrated by Echegoyen et al. (Moonen, 2000; Beulen, 2000). Addition of excess electrons (via exhaustive reduction) to methanofullerene derivatives regenerates underivatized fullerenes. Addition of less than an excess amount of electrons induces only a substituent migration or a “walk-on-the-sphere” isomerization of the methanofullerene groups (Kessinger, 1998).
There are a number of additional reports of methods for derivatization of fullerenes. Gugel, U.S. Pat. No. 5,763,719, reports methods for making thermally stable fullerenes. Murphy et. al., U.S. Pat. No. 6,162,926 and PCT published application WO9636631, report methods for making multiply-substituted fullerenes. Cahill and Henderson, U.S. Pat. No. 5,475,172, report methods of functionalizing fullerenes employing organoborane intermediates. Mattay et. al., PCT published application WO 9626186, reports methods for the generation of azafullerenes. Hinokuma et. al., EP 1 071149, reports the introduction of one or more proton releasable groups such as OH and SO3H into fullerenes. Kampe et. al., U.S. Pat. No. 5,587,476 and PCT published application WO 9405671, report fullerene derivatives prepared by reaction with diamines. Chiang, U.S. Pat. Nos. 5,648,523; 5,994,410; 6,020,523 and 6,046,361, reports the preparation of fullerene derivatives including polyorganofullerenes and water-soluble fullerene derivatives. Friedman et. al., U.S. Pat. Nos. 5,811,460 and 6,204,391, report water-soluble fullerene derivatives. Schriver et. al., U.S. Pat. No. 5,503,643, reports fuel oil-substituted fullerenes.
Endohedral fullerenes are those fullerenes encapsulating an atom or atoms in their hollow interior spaces. They are written with the general formula Mm@C2n, where M is an element, m is the integer 1, 2, 3 or higher, and n is an integer number. The “@” symbol refers to the endohedral or interior nature of the M atom inside of the fullerene cage. Aspects of endohedral metallofullerenes have been reviewed (Bethune, 1993; Nagase, 1996; Nagase, 2000; Shinohara, 2000; Liu, 2000).
Endohedral fullerenes corresponding to most of the empty fullerene cages have been produced and detected under varied conditions. Because the endohedral element is completely encased by the spherical fullerene shell, it is not released to the exterior except under cage-destructive conditions (heat, long-duration exposure to strong acids, etc.). Endohedrals containing lanthanide, transition, alkali, and alkaline earth metals have by far received the most attention to date, although studies on non-metallo endohedrals such as He@C60, N@C60, P@C60, etc. are developing. The major production technique for producing endohedral metallofullerenes is the Krätschmer-Huffman style resistive heating of graphite rods that are impregnated with metals or metal salts. Typically, metallofullerenes comprise only several percent of the total fullerene yield in the carbon arc production method.
Of the Mm@C2n metallo-endohedrals, those having the particular formulation M@C82 (with M=a lanthanide element) have been the subjects of most investigations to date. This is largely due to the solubility of the M@C82 closed-shell species in common solvents. Because of their solubility, they can be separated from the empty fullerenes and other endohedral metallofullerenes, including their different cage isomers, by chromatographic methods. Their solubility also allows for standard derivatizations to be performed on the metallo-endohedral fullerenes, by analogy to the derivation of empty large HOMO-LUMO gap fullerenes.
The study of other M@C2n, and multiple-metal containing Mm@C2n materials is proceeding at more slowly because many of these fullerenes are intrinsically insoluble in the common, non-reactive hydrocarbon and arene solvents normally used for C60, etc. Without wishing to be bound by any particular theory, the most likely explanation for their insolubility is the spontaneous formation of intramolecular polymers.
The “polymerization” which occurs for certain metallofullerenes, most notably the M@C60 species, derives from their electronic structures. The electronic structure of a metallofullerene of course encompasses the interior metal and any electrons it donates to the fullerene cage molecular orbital. Essentially, these fullerenes have small HOMO-LUMO or band gaps such that the molecules are open-shell or very close in energy to being open shell. Functionally equivalent descriptions to this situation include the descriptions that metallofullerenes possess “radical sites”, “dangling bonds”, or “unfulfilled vacancies”. Most endohedral metallofullerenes have been found to contain metals which donate three electrons (per metal) forming endohedral M3+ ions inside of a fullerene cage now having three “extra” electrons relative to a cage without a metal inside. Such an endohedral metallofullerene molecule is a zwitterion, with a cationic metal “core” and anionic fullerene (fulleride) cage.
Because the number of electrons transferred to the fullerene cage (three) is an odd number, there has to be an unpaired electron, i.e. the molecule is a radical. As many radicals do, they dimerize or oligomerize spontaneously “quenching” their free radical status. To free them from their intramolecularly cross-linked matrix, their electronic structures must be altered. To eliminate radical behavior, redox chemistry (addition or removal of molecular electron(s)) can be performed, or they can be derivatized exohedrally by substituent groups. It should be pointed out that the matrix of “polymerized” insoluble endohedral fullerenes likely contains some small percentage of soluble C2n species interstitially trapped and/or bonded to other endohedral fullerenes, as a consequence of heterogeneous nature of fullerene generation in the arc process.
Despite the relatively low availability of samples of endohedral fullerenes, their derivatization chemistry is also proceeding (Kato, 1997). Examples include the derivatization of the soluble endohedral metallofullerenes M@C82 (M=La, Gd), La2@C80, and Sc2@C84 with disiliranes and digermanes (Akasaka, 1995(a); Akasaka, 1995(b); Akasaka, 1995(c); Yamamoto, 1999). Suzuki and co-workers reported the reaction of La@C82 with substituted carbenes derived from diazomethanes to produce cyclopropanated derivatives (Suzuki, 1995; Yamamoto, 1998). Various groups have reported the polyhydroxylation of endohedral metallofullerenes including Ho@C82 (Cagle, 1999), Ho2@C82 (Cagle, 1999), Pr@C82 (Sun, 1999), and Gd@C82 (Wilson, 1999; Mikawa, 2001).
Patents involving the exohedral derivatization of endohedral metallofullerenes include U.S. Pat. No. 5,717,076 (Yamamoto, 1998) and U.S. Pat. No. 5,869,626 (Yamamoto, 1999). The first involves cyclopropanations conducted in a completely different manner than Bingel-style reactions and adding four or fewer groups to the fullerene surface, while the second is an unrelated type of derivatization.
Bingel-style cyclopropanations have not been reported on endohedral metallofullerenes. Bingel-style cyclopropanations have been reported on minute amounts of “inert” endohedral fullerenes present in trace levels (10−4 M or 100 ppm) in C60 matrices, including N@C60 (Dietel, 1999), 3He@C60 (Cross, 1996) (and 3H@C60 (Khong, 2000)). These materials were produced for EPR and 3He-NMR detection purposes. Both techniques are very sensitive to trace amounts of spectroscopically active material present in a matrix of dominantly unactive material. Neither has radical character expressed by the fullerene cage, explaining their apparent solubility and lack of difference in reactivity for Bigel-style cyclopropanation from that of C60.
Reaction of M@C60 (and M@C70, etc.) with nucleophilic solvents to extract soluble surface-modified (normally insoluble) M@C2n species has been demonstrated with the nitrogen-bases aniline and pyridine. Examples of solvents used and endohedral fullerenes extracted include: aniline for M@C60 (M=Y, Ba, La, Ce, Pr, Nd, Gd; Kubozono, 1996(b)), aniline for Eu@C60 (Inoue, 2000), aniline for Er@C60 (Ogawa, 2000), aniline for Ca@C60 and Sr@C60 (Kubozono, 1996(a)), pyridine for Ca@C60 (Kubozono, 1995), and pyridine for mixed M@C2n (M=La, Ce; Liu 1998). These highly nucleophilic solvents, with strongly coordinating amine nitrogen groups, may irreversibly produce soluble, poly-solvent coordinated species. The chemical nature of the surface attachment has not been analytically determined; a covalent linkage is most likely. The formation of adducts may proceed via charge-transfer, donor-acceptor complexes, and/or reduced fullerene species prior to covalent bond formation (Skiebe, 1994; Klos, 1994). In general, the solvent molecules in these cases cannot be removed from bonding to the fullerene cages, for example by applying reduced pressure and/or non-destructive heat.
Gügel, Müllen and co-workers reported the extraction of giant fullerenes from arc-produced fullerene soot by an irreversible Diels-Alder cycloaddition strategy using ortho-quinodimethanes (Beer et. al.,1997). These highly reactive intermediates, obtained by thermal extrusion of sulfur dioxide from the requisite organic thiophenes, add irreversibly across fullerene carbon-carbon double bonds. Other reports on giant fullerene and SBF solubilizations include the following. There are several reports of solvent extraction of “giant” fullerenes C2n (n≧50) with high-boiling arene solvents (Parker, 1991; Diederich, 1991). Yeretzian et. al. conducted a study of gradient sublimation on empty and endohedral metallofullerenes, showing that the technique could be used to generate a partial enrichment of the larger (empty) C2n (n≧47) fullerenes without solvents (Yeretzian, 1993). The SBF C74 specifically was solubilized by electrochemical one-electron reduction, which was used to separate it from the more numerous large-bandgap fullerenes (Diener, 1998).
The medicinal applications of fullerene compounds have been reviewed by several authors (Wilson, 1999; Wilson, 2000(a)). Watson et. al. mention use of endohedral fullerenes in applications like medical imaging in their U.S. patent (Watson, 1997), but provide few details on how the application would be achieved. Gd chelates as MRI contrast agents have been thoroughly reviewed by Caravan and others (Caravan, 1999). Zhang (1997), Wilson (1999), and Mikawa (2001) reported aqueous relaxivity measurements on Gd@C82(OH)x, showing higher R1 values than obtained with conventional inorganic chelates. Neutron activation of 165Hom@C2n to 166Hom@C2n with a biodistribution study was reported (Cagle, 1996; Cagle, 1999). Dugan and co-workers have reported medicinal antioxidant and other properties of “Bingelated” C60 (Dugan, 2000).
There remains a need in the art for improved methods of derivatization and/or solubilization of fullerenes, especially of non-derivatized and derivatized fullerenic species that are insoluble or substantially insoluble in polar aprotic solvents.